Apoptosis or programmed cell death is an essential physiological process of selective elimination of cells in multicellular organisms. This process is invoked during normal organ development and tissue homeostasis and also during certain pathological conditions that result in degenerative diseases. Programmed cell death plays an important role in regulating the multiplication of and pathogenesis by a number of viruses. In virus-infected cells, apoptotic paradigms are initiated as a cellular defensive mechanism to eliminate infected cells. However, a number of viruses encode proteins that suppress apoptosis resulting in efficient viral replication and pathogenesis. Human adenovinises have evolved multiple strategies employing proteins coded by three different gene blocks, E1B, E3, and E4 to overcome the effect of apoptosis of infected permissive cells. One of the proteins coded by the E1B region, the 19K protein confers a survival fiction in adenovirus-infected cells and prevents premature cell death. Adenovirus mutants defective in the 19K gene produced large clear plaques on infected cell monolayers. Several of these mutants induce an enhanced cytopathic effect in infected cells resulting in cellular destruction as well as fragmentation of cellular and viral DNA. The DNA fragmentation induced by 19K mutants is reminiscent of that observed during apoptosis. Although it has not yet been determined whether DNA fragmentation induced by 19K mutants occurs by an apoptotic mechanism, it is clear that the 19 kDa protein protects against a cell death program induced by viral infection, thus facilitating efficient virus replication. In addition, the 19 kDa protein suppresses the cytotoxic effects of certain external stimuli such as the tumor necrosis factor xcex1 and anti-Fas antibody. Both of these agents cause cell death through apoptosis. Similarly, the 19 kDa protein protects cells against the effects of DNA-damaging agents such as the anti-cancer drug cisplatin and ultraviolet radiation induced cell death through a p53-dependent apoptotic pathway. Cells expressing the 19 kDa protein efficiently suppress cell death induced by overexpression of p53. Thus, the 19 kDa protein provides a survival function in virus-infected cells and also protects cells against certain other death-inducing stimuli.
The survival function provided by E1B 19K is similar to the activity of the cellular protooncogene Bcl-2. The Bcl-2 oncogene was isolated from a follicular lymphoma and has been shown to enhance the survival of hematopoietic B and T cells by blocking apoptosis. In addition, overexpression of Bcl-2 protein inhibits apoptosis induced by exposure to diverse stimuli possibly through different pathways. Although the effect of 19 kDa protein expression on cell death induced by diverse stimuli has not been extensively examined, the Bcl-2 protein can clearly substitute for the 19 kDa protein during adenovirus infection. Characteristic fragmentation of cellular DNA induced by infection with adenovirus type 2 (Ad2) 19K mutants is efficiently suppressed in cells ectopically expressing the human Bcl-2 protein. Similarly, expression of Bcl-2 by an Ad2-Bcl-2 recombinant virus (Ad-Bcl2) can fully substitute for the 19K function. The Ad-Bcl12 virus does not induce DNA fragmentation in infected cells and forms small plaques on cell monolayers. Rao et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89:7742-46 have reported that Bcl-2 can substitute for 19K in transformation of primary rat kidney cells in cooperation with E1A. Although these results do not clarify whether the 19 kDa and Bcl-2 proteins function by similar mechanisms, they indicate that these two proteins can provide cell survival function against certain stimuli.
The mechanism by which the 19K gene and the Bcl-2 protooncogene protect against cell death is not known. These proteins may mediate cell survival by interacting with certain cellular proteins. Boyd J. M. et al. (1994) Cell 79:341-351, have identified three proteins, NIP1, NIP2, and NIP3, which interact with both the adenovirus E1B 19 kDa protein and the Bcl-2 protein. The NIP1, NIP2, and NIP3 proteins are believed to play a role in the ability of the E1B 19 kDa protein to provide a cell survival function.
The present invention is based, at least in part, on the discovery of novel NIP2 family members, referred to herein as xe2x80x9cNIP2b, NIP2cL, and NIP2cSxe2x80x9d nucleic acid and protein molecules. The NIP2b, NIP2cL, and NIP2cS molecules of the present invention are useful as modulating agents for regulating a variety of cellular processes. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding NIP2b, NIP2cL, and NIP2cS proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of NIP2b, NIP2cL, and NIP2cS-encoding nucleic acids.
In one embodiment, a NIP2b, NIP2cL, or NIP2cS nucleic acid molecule of the invention is at least 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or more identical to the nucleotide sequence (e.g., to the entire length of the nucleotide sequence) shown in SEQ ID NO:1, 3, 4, 6, 7, or 9, or a complement thereof.
In a preferred embodiment, the isolated nucleic acid molecule includes the nucleotide sequence shown SEQ ID NO:1 or 3, or a complement thereof. In another embodiment, the nucleic acid molecule includes SEQ ID NO:3 and nucleotides 1-369 of SEQ ID NO: 1. In another embodiment, the nucleic acid molecule includes SEQ ID NO:3 and nucleotides 1483-3076 of SEQ ID NO:1. In another preferred embodiment, the nucleic acid molecule consists of the nucleotide sequence shown in SEQ ID NO:1 or 3. In another preferred embodiment, the nucleic acid molecule includes a fragment of at least 535 nucleotides (e.g., 535 contiguous nucleotides) of the nucleotide sequence of SEQ ID NO:1 or 3, or a complement thereof.
In another preferred embodiment, the isolated nucleic acid molecule includes the nucleotide sequence shown SEQ ID NO:4 or 6, or a complement thereof. In another embodiment, the nucleic acid molecule includes SEQ ID NO:6 and nucleotides 1-22 of SEQ ID NO:4. In another embodiment, the nucleic acid molecule includes SEQ ID NO:6 and nucleotides 989-4235 of SEQ ID NO:4. In another preferred embodiment, the nucleic acid molecule consists of the nucleotide sequence shown in SEQ ID NO:4 or 6. In another preferred embodiment, the nucleic acid molecule includes a fragment of at least 3225 nucleotides (e.g., 3225 contiguous nucleotides) of the nucleotide sequence of SEQ ID NO:4, SEQ ID NO:6, or a complement thereof.
In a preferred embodiment, the isolated nucleic acid molecule includes the nucleotide sequence shown SEQ ID NO:7 or 9, or a complement thereof. In another embodiment, the nucleic acid molecule includes SEQ ID NO:9 and nucleotides 1-55 of SEQ ID NO:7. In another embodiment, the nucleic acid molecule includes SEQ ID NO:9 and nucleotides 434-2966 of SEQ ID NO:7. In another preferred embodiment, the nucleic acid molecule consists of the nucleotide sequence shown in SEQ ID NO:7 or 9. In another preferred embodiment, the nucleic acid molecule includes a fragment of at least 461 nucleotides (e.g., 461 contiguous nucleotides) of the nucleotide sequence of SEQ ID NO:7, SEQ ID NO:9, or a complement thereof.
In another embodiment, a NIP2b, NIP2cL, and NIP2cS nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO:2, 5, or 8. In a preferred embodiment, a NIP2b, NIP2cL, and NIP2cS nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to the entire length of the amino acid sequence of SEQ ID NO:2, 5, or 8.
In another preferred embodiment, an isolated nucleic acid molecule encodes the amino acid sequence of human NIP2b, NIP2cL, and NIP2cS. In yet another preferred embodiment, the nucleic acid molecule includes a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO:2, 5, or 8. In yet another preferred embodiment, the nucleic acid molecule is at least 461, 535, or 3225 nucleotides in length. In a further preferred embodiment, the nucleic acid molecule is at least 461, 535, or 3225 nucleotides in length and encodes a protein having a NIP2b, NIP2cL, and NIP2cS activity (as described herein).
Another embodiment of the invention features nucleic acid molecules, preferably NIP2b, NIP2cL, and NIP2cS nucleic acid molecules, which specifically detect NIP2b, NIP2cL, and NIP2cS nucleic acid molecules relative to nucleic acid molecules encoding non-NIP2b, non-NIP2cL, and non-NIP2cS proteins. For example, in one embodiment, such a nucleic acid molecule is at least 300-350, 350-400, 400-450, 461, 461-500, 535, 535-600 or more nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:1, 4, or 7, or a complement thereof.
In preferred embodiments, the nucleic acid molecules are at least 15 (e.g., contiguous) nucleotides in length and hybridize under stringent conditions to nucleotides 1-348, 431-478, 676-784, 1538-1546, 1587-1598, 1615-1727, or 2070-3076 of SEQ ID NO:1. In other preferred embodiments, the nucleic acid molecules comprise nucleotides 1-348, 431-478, 676-784, 1538-1546, 1587-1598, 1615-1727, or 2070-3076 of SEQ ID NO:1.
In other preferred embodiments, the nucleic acid molecules are at least 15 (e.g., contiguous) nucleotides in length and hybridize under stringent conditions to nucleotides 1-16 of SEQ ID NO:4. In other preferred embodiments, the nucleic acid molecules comprise nucleotides 1-16 of SEQ ID NO:4.
In yet other preferred embodiments, the nucleic acid molecules are at least 15 (e.g., contiguous) nucleotides in length and hybridize under stringent conditions to nucleotides 1-26, 406-598, 1058-2703, 2775-2936, or 2956-2966 of SEQ ID NO:7. In other preferred embodiments, the nucleic acid molecules comprise nucleotides 1-26, 406-598, 1058-2703, 2775-2936, or 2956-2966 of SEQ ID NO:7.
In other preferred embodiments, the nucleic acid molecule encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, 5, or 8, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO:1, 3, 4, 6, 7, or 9 under stringent conditions.
Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to a NIP2b, NIP2cL, and NIP2cS nucleic acid molecule, e.g., the coding strand of a NIP2b, NIP2cL, and NIP2cS nucleic acid molecule.
Another aspect of the invention provides a vector comprising a NIP2b, a NIP2cL, or a NIP2cS nucleic acid molecule. In certain embodiments, the vector is a recombinant expression vector. In another embodiment, the invention provides a host cell containing a vector of the invention. In yet another embodiment, the invention provides a host cell containing a nucleic acid molecule of the invention. The invention also provides a method for producing a protein, preferably a NIP2b, NIP2cL, and NIP2cS protein, by culturing in a suitable medium, a host cell, e.g., a mammalian host cell such as a non-human mammalian cell, of the invention containing a recombinant expression vector, such that the protein is produced.
Another aspect of this invention features isolated or recombinant NIP2b, NIP2cL, and NIP2cS proteins and polypeptides. In one embodiment, the isolated protein, preferably a NIP2b, NIP2cL, or NIP2cS protein, includes at least one transmembrane domain. In a preferred embodiments, the isolated protein, preferably a NIP2b, NIP2cL, or NIP2cS protein, includes at least one calcium binding domain. In yet another preferred embodiment, the isolated protein, preferably a NIP2b, NIP2cL, or NIP2cS protein, includes at least one xe2x80x9c4 transmembrane segment integral membrane protein domainxe2x80x9d. In a preferred embodiment, the protein, preferably a NIP2b, NIP2cL, or NIP2cS protein, includes at least one transmembrane domain and has an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to the amino acid sequence of SEQ ID NO:2, 5, or 8. In another embodiment, the protein, preferably a NIP2b, NIP2cL, or NIP2cS protein, includes at least one calcium-binding domain and has an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to the amino acid sequence of SEQ ID NO:2, 5, or 8. In yet another preferred embodiment, the protein, preferably a NIP2b, NIP2cL, or NIP2cS protein, includes at least one xe2x80x9c4 transmembrane segment integral membrane protein domainxe2x80x9d and has an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to the amino acid sequence of SEQ ID NO:2, 5, or 8. In another preferred embodiment, the protein, preferably a NIP2b, NIP2cL, or NIP2cS protein, includes at least one transmembrane domain and plays a role in apoptosis or programmed cell death. In another embodiment, the protein, preferably a NIP2b, NIP2cL, or NIP2cS protein, includes at least one calcium-binding domain and plays a role in apoptosis or programmed cell death. In yet another preferred embodiment, the protein, preferably a NIP2b, NIP2cL, or NIP2cS protein, includes at least one xe2x80x9c4 transmembrane segment integral membrane protein domainxe2x80x9d and plays a role in apoptosis or programmed cell death. In yet another preferred embodiment, the protein, preferably a NIP2b, NIP2cL, and NIP2cS protein, includes at least one transmembrane domain and is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, 3, 4, 6, 7, or 9. In another embodiment, the protein, preferably a NIP2b, NIP2cl, or NIP2cS protein, includes at least one calcium-binding domain and is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, 3, 4, 6, 7, or 9. In yet another embodiment, the protein, preferably a NIP2b, NIP2cL, or NIP2cS protein, includes at least one xe2x80x9c4 transmembrane segment integral membrane protein domainxe2x80x9d and is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, 3, 4, 6, 7, or 9.
In another embodiment, the invention features fragments of the protein having the amino acid sequence of SEQ ID NO:2, 5, or 8, wherein the fragment comprises at least 15 amino acids (e.g., contiguous amino acids) of the amino acid sequence of SEQ ID NO:2, 5, or 8. In another embodiment, the protein, preferably a NIP2b, NIP2cL, or NIP2cS protein, has the amino acid sequence of SEQ ID NO:2, 5, or 8, respectively.
In another embodiment, the invention features an isolated protein, preferably a NIP2b, NIP2cL, and NIP2cS protein, which is encoded by a nucleic acid molecule consisting of a nucleotide sequence at least about 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to a nucleotide sequence of SEQ ID NO:1, 3, 4, 6, 7, or 9, or a complement thereof. This invention further features an isolated protein, preferably a NIP2b, NIP2cL, or NIP2cS protein, which is encoded by a nucleic acid molecule consisting of a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1, 3, 4, 6, 7, or 9, or a complement thereof.
The proteins of the present invention or portions thereof, e.g., biologically active portions thereof, can be operatively linked to a non-NIP2b, non-NIP2cL, or non-NIP2cS polypeptide (e.g., heterologous amino acid sequences) to form fusion proteins. The invention further features antibodies, such as monoclonal or polyclonal antibodies, that specifically bind proteins of the invention, preferably NIP2b, NIP2cL, and NIP2cS proteins. In addition, the NIP2b, NIP2cL, and NIP2cS proteins or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.
In another aspect, the present invention provides a method for detecting the presence of a NIP2b, NIP2cL, and NIP2cS nucleic acid molecule, protein or polypeptide in a biological sample by contacting the biological sample with an agent capable of detecting a NIP2b, NIP2cL, and NIP2cS nucleic acid molecule, protein or polypeptide such that the presence of a NIP2b, NIP2cL, and NIP2cS nucleic acid molecule, protein or polypeptide is detected in the biological sample.
In another aspect, the present invention provides a method for detecting the presence of NIP2b, NIP2cL, and NIP2cS activity in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of NIP2b, NIP2cL, and NIP2cS activity such that the presence of NIP2b, NIP2cL, and NIP2cS activity is detected in the biological sample.
In another aspect, the invention provides a method for modulating NIP2b, NIP2cL, and NIP2cS activity comprising contacting a cell capable of expressing NIP2b, NIP2cL, and NIP2cS with an agent that modulates NIP2b, NIP2cL, and NIP2cS activity such that NIP2b, NIP2cL, and NIP2cS activity in the cell is modulated. In one embodiment, the agent inhibits NIP2b, NIP2cL, and NIP2cS activity. In another embodiment, the agent stimulates NIP2b, NIP2cL, and NIP2cS activity. In one embodiment, the agent is an antibody that specifically binds to a NIP2b, NIP2cL, and NIP2cS protein. In another embodiment, the agent modulates expression of NIP2b, NIP2cL, and NIP2cS by modulating transcription of a NIP2b, NIP2cL, and NIP2cS gene or translation of a NIP2b, NIP2cL, and NIP2cS mRNA. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of a NIP2b, NIP2cL, and NIP2cS mRNA or a NIP2b, NIP2cL, and NIP2cS gene.
In one embodiment, the methods of the present invention are used to treat a subject having a disorder characterized by aberrant NIP2b, NIP2cL, and NIP2cS protein or nucleic acid expression or activity by administering an agent which is a NIP2b, NIP2cL, and NIP2cS modulator to the subject. In one embodiment, the NIP2b, NIP2cL, and NIP2cS modulator is a NIP2b, NIP2cL, and NIP2cS protein. In another embodiment the NIP2b, NIP2cL, and NIP2cS modulator is a NIP2b, NIP2cL, and NIP2cS nucleic acid molecule. In yet another embodiment, the NIP2b, NIP2cL, and NIP2cS modulator is a peptide, peptidomimetic, or other small molecule. In a preferred embodiment, the disorder characterized by aberrant NIP2b, NIP2cL, and NIP2cS protein or nucleic acid expression is a disorder characterized by deregulated programmed cell death.
The present invention also provides a diagnostic assay for identifying the presence or absence of a genetic alteration characterized by at least one of (i) aberrant modification or mutation of a gene encoding a NIP2b, NIP2cL, and NIP2cS protein; (ii) mis-regulation of the gene; and (iii) aberrant post-translational modification of a NIP2b, NIP2cL, and NIP2cS protein, wherein a wild-type form of the gene encodes a protein with a NIP2b, NIP2cL, and NIP2cS activity.
In another aspect the invention provides a method for identifying a compound that binds to or modulates the activity of a NIP2b, NIP2cL, and NIP2cS protein, by providing an indicator composition comprising a NIP2b, NIP2cL, and NIP2cS protein having NIP2b, NIP2cL, and NIP2cS activity, contacting the indicator composition with a test compound, and determining the effect of the test compound on NIP2b, NIP2cL, and NIP2cS activity in the indicator composition to identify a compound that modulates the activity of a NIP2b, NIP2cL, and NIP2cS protein.88
Other features and advantages of the invention will be apparent from the following detailed description and claims.